The invention relates to human, humanized and monoclonal antibodies with decreased immunogenicity, uses and methods of production thereof. The invention in particular relates to antagonistic anti-human CD40 monoclonal antibodies.
The CD40 molecule is a 50 kDa type I membrane glycoprotein and is expressed on B cells, monocytes/macrophages, dendritic cells (DCs) and activated endothelial cells.1-6 Under certain conditions, CD40 can also be found on fibroblasts, epithelial cells and keratinocytes.7 CD40 ligand (CD40L, CD154), a 32 kDa type II integral membrane glycoprotein, is transiently expressed on activated CD4+ T cells and a small population of activated CD8+ T cells.8, 9 In addition, CD40L has been found on a number of other cell types after activation, including mast cells, basophils, B cells, eosinophils, DCs and platelets.10, 11 
Studies in murine models have clearly demonstrated the involvement of the CD40L-CD40 interaction in the pathophysiology of various autoimmune diseases (for review, see reference12). Evidence from CD40L transgenic mice, which acquire lethal inflammatory bowel disease, provided the first evidence that CD40-CD40L interactions might also play a role in the pathogenesis of inflammatory bowel diseases.13 An anti-mouse CD40L monoclonal antibody (Mab) effectively prevents mucosal inflammation and interferon-γ production by lamina propria CD4+ T cells in TNBS-induced colitis.14 In a Severe Combined Immunodeficiency (SCID) mouse inflammatory bowel disease model it was shown that treatment with anti-CD40L from the day of T-cell reconstitution completely prevented clinical and histological appearance of experimental colitis.15 Furthermore, anti-CD40L administration from week 5 after T-cell reconstitution could still prevent progression of the disease and treated animals showed improvement in disease symptoms and histology compared with control animals.15 In addition, reconstitution of SCID mice with T cells from CD40L knock-out mice, further demonstrated the essential role of CD40L-expressing T cells in disease development and interleukin-12 production.16 
The CD40-CD40L interaction can be antagonized with monoclonal antibodies (Mabs) against either CD40L or CD40. The expression of CD40L on activated platelets has resulted in thrombo-embolic events during treatment of humans with IgG1 anti-human CD40L Mabs at higher dose levels and termination of the development of these Mabs.17, 19 Antagonizing CD40 therefore seems a more attractive approach. The non-stimulatory antagonistic activity of Mab 5D12 (anti-human CD40) was demonstrated in various in vitro studies using different CD40-bearing cell types20, 22 and chimeric 5D12 (ch5D12) antagonist activity was validated in vivo using various non-human primate disease models.23, 27 ch5D12 is a molecularly engineered human IgG4 antibody containing the murine variable domains of the heavy and light chains of 5D12 and was constructed to reduce the potential for immunogenicity and to enhance the in vivo half-life of the murine 5D12 Mab when used in humans.
Patients with Crohn's disease suffer from a debilitating inflammatory disorder of the gastrointestinal tract of which the precise aetiology and pathogenesis remain elusive.28, 29 The disease is characterized by an influx into diseased mucosa of activated T cells, B cells and macrophages,30, 31 local production of soluble mediators of inflammation, and damage of involved tissues.28, 29 Mucosal CD4+ T cells and macrophages and cytokines such as tumour necrosis factor (TNF)-α and IL-12 have been shown to play a central role in initiating an inflammatory loop in Crohn's disease.32, 38 T cells from inflamed mucosa exhibit a higher proliferative capacity,28, 29 and secrete increased amounts of IFN-γ and IL-2. Increased levels of T-cell associated cytokine mRNA transcripts have been found in mucosal biopsies from Crohn's disease patients.33 A dominant role of CD40L on the activated CD4+ T cells has been suggested in our studies on CD40/CD40L expression in Crohn's disease lesions.39 CD40L can mediate a strong activation of CD40-bearing cells, predominantly B cells and macrophages, thus resulting in increased production of TNF-α and IL-12 in lesions. Using immunohistochemistry, increased staining with 5D12 was found in all samples of diseased areas from Crohn's disease patients compared to non-diseased areas. Double staining for CD40 and CD20 (B cells) or CD68 (macrophages) indicated that in the sections from patients with Crohn's disease, CD40+ cells were mainly B cells in the lymphoid follicles and macrophages in the lamina propria. Lamina propria T cells from inflamed mucosa of Crohn's disease patients induced monocytes to produce significant amounts of IL-12 and TNF-α after 48 h of co-culture. Addition of 5D12 resulted in reduced IL-12 and TNF-α production; levels of production were reduced to the levels observed using control lamina propria T cells both in the absence and presence of IFN-γ.39 